Epigallocatechin gallate delays age-related cataract development via the RASSF2/AKT pathway.

Age-related cataract (ARC) is a common eye disease, the main cause of which is oxidative stress-mediated apoptosis of lens epithelial cells (LECs). Epigallocatechin gallate (EGCG) is the most potent antioxidant in green tea. Our results demonstrated that EGCG could effectively reduce apoptosis of LECs and retard lens clouding in aged mice. By comparing transcriptome sequencing results of three groups of mice (young control, untreated aged, and EGCG-treated) and screening using GO and KEGG analyses, we selected RASSF2 as the effector gene of EGCG for mechanistic exploration. We verified that the differential expression of RASSF2 was associated with the occurrence of ARC in clinical samples and mouse tissues by immunohistochemistry and western blotting, respectively. We showed that high RASSF2 expression plays a crucial role in the oxidative induction of apoptosis in LECs, as revealed by overexpression and interference experiments. Further studies showed that RASSF2 mediates the inhibitory effect of EGCG on apoptosis and ARCogenesis in LECs by regulating AKT (Ser473) phosphorylation. In this study, we found for the first time the retarding effect of EGCG on lens clouding in mice and revealed the mechanism of action of RASSF2/AKT in it, which provides a theoretical basis for the targeted treatment of EGCG.

SOCS1 as a Biomarker Candidate for HPV Infection and Prognosis of Head and Neck Squamous Cell Carcinomas.

The pathogenesis of head and neck squamous cell carcinoma (HNSCC) is associated with human papillomavirus (HPV) infection. However, the molecular mechanisms underlying the interactions between HNSCC and HPV remain unclear. Bioinformatics was used to analyze the gene expression dataset of HPV-associated HNSCC based on the Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) in HPV-positive and HPV-negative HNSCC were screened. Gene function enrichment, protein-protein interactions (PPI), survival analysis, and immune cell infiltration of DEGs were performed. Furthermore, the clinical data of HNSCC tissue samples were analyzed using immunohistochemistry. In total, 194 DEGs were identified. A PPI network was constructed and 10 hub genes (EREG, PLCG1, ERBB4, HBEGF, ZFP42, CBX6, NFKBIA, SOCS1, ATP2B2, and CEND1) were identified. Survival analysis indicated that low expression of SOCS1 was associated with worse overall survival. Immunohistochemistry demonstrated that SOCS1 expression was higher in HPV-negative HNSCC than in HPV-positive HNSCC, and there was a positive correlation between SOCS1 expression and patient survival. This study provides new information on biological targets that may be relevant to the molecular mechanisms underpinning the occurrence and development of HNSCC. SOCS1 may play an important role in the interaction between HPV and HNSCC and serve as a potential biomarker for future therapeutic targets.

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis.

BACKGROUND: Cotton has tremendous economic value worldwide; however, its allopolyploid nature and time-consuming transformation methods have hampered the development of cotton functional genomics. The protoplast system has proven to be an important and versatile tool for functional genomics, tissue-specific marker gene identification, tracking developmental trajectories, and genome editing in plants. Nevertheless, the isolation of abundant viable protoplasts suitable for single-cell RNA sequencing (scRNA-seq) and genome editing remains a challenge in cotton. RESULTS: We established an efficient transient gene expression system using protoplasts isolated from cotton taproots. The system enables the isolation of large numbers of viable protoplasts and uses an optimized PEG-mediated transfection protocol. The highest yield (3.55 × 105/g) and viability (93.3%) of protoplasts were obtained from cotton roots grown in hydroponics for 72 h. The protoplasts isolated were suitable for scRNA-seq. The highest transfection efficiency (80%) was achieved when protoplasts were isolated as described above and transfected with 20 µg of plasmid for 20 min in a solution containing 200 mM Ca2+. Our protoplast-based transient expression system is suitable for various applications, including validation the efficiency of CRISPR vectors, protein subcellular localization analysis, and protein-protein interaction studies. CONCLUSIONS: The protoplast isolation and transfection protocol developed in this study is stable, versatile, and time-saving. It will accelerate functional genomics and molecular breeding in cotton.

Virus-Induced Gene Editing and Its Applications in Plants.

CRISPR/Cas-based genome editing technologies, which allow the precise manipulation of plant genomes, have revolutionized plant science and enabled the creation of germplasms with beneficial traits. In order to apply these technologies, CRISPR/Cas reagents must be delivered into plant cells; however, this is limited by tissue culture challenges. Recently, viral vectors have been used to deliver CRISPR/Cas reagents into plant cells. Virus-induced genome editing (VIGE) has emerged as a powerful method with several advantages, including high editing efficiency and a simplified process for generating gene-edited DNA-free plants. Here, we briefly describe CRISPR/Cas-based genome editing. We then focus on VIGE systems and the types of viruses used currently for CRISPR/Cas9 cassette delivery and genome editing. We also highlight recent applications of and advances in VIGE in plants. Finally, we discuss the challenges and potential for VIGE in plants.

High MST2 expression regulates lens epithelial cell apoptosis in age-related cataracts through YAP1 targeting GLUT1.

Age-related cataract (ARC) is a severe visual impairment disease and its pathogenesis remains unclear. This study investigated the relevance of MST2/YAP1/GLUT1 in ARC development in vivo and in vitro, and explored the role and possible mechanisms of this pathway in oxidative damage-mediated apoptosis of lens epithelial cells (LECs). Western blot analysis and immunohistochemistry showed that MST2 and phosphorylated (p)-YAP (Ser127) protein levels were increased, and YAP1 and GLUT1 protein levels were decreased in LECs of ARC patients and aged mice. Additionally, differential expression of MST2 and YAP1 was associated with H2O2-induced apoptosis of human lens epithelial B3 (HLE-B3) cells. CCK-8 and Hoechst 33,342 apoptosis assays showed that MST2 and YAP1 were involved in H2O2-induced apoptosis of LECs. Subsequent experiments showed that, during MST2-mediated H2O2-induced apoptosis, p-YAP (Ser127) levels were elevated and immunofluorescence revealed nucleoplasmic translocation and inhibition of YAP1 protein expression. Furthermore, GLUT1 was in turn synergistically transcriptionally regulated by YAP1-TEAD1 in dual luciferase reporter assays. In conclusion, our study indicates that the MST2/YAP1/GLUT1 pathway plays a major role in the pathogenesis of ARC and LECs apoptosis, providing a new direction for future development of targeted inhibitors that block this signaling pathway to prevent, delay, or even cure ARC.

Changes in gene expression in rat placenta at gestational day 16.5 in response to hyperglycemia.

Gestational diabetes mellitus (GDM) is a serious pregnancy complication. Hyperglycemia induces abnormal placental development and function. However, the mechanism is unclear. Previous research showed streptozocin (STZ) injection sustained hyperglycemia throughout pregnancy in rodents. Our current results showed that the placenta from hyperglycemic STZ-treated rats was about 20% heavier than that of controls. The relative thickness of each layer of the placenta was also significantly different on gestational day (GD) 16.5. Gene expression was analyzed by RNA sequencing to explore reasons for the abnormal placenta. In total, 2100 differential expressed genes (DEGs), including 1327 up-regulated and 773 down-regulated genes, were identified. Gene ontogeny (GO) analysis revealed DEGs involved in developmental process, growth, metabolic process, cell junction, molecular transducer activity and signaling. By KEGG analysis, DEGs were mainly related to the endocrine system, development, signal transduction and cell growth and death. The KEGG results were partly consistent with GO results, with DEGs mainly focused on biochemical signal pathways such as cell growth and death (e.g., Abl1, Bbc3 and Camk2d), and signal transduction (e.g., Abl1, Ceacam1 and Arnt). These genes may play a dominant role in abnormal cell proliferation and signaling disorders. These results suggest that DEGs play a role in diabetic-induced placental abnormalities. One or more of these DEGs may be involved in the etiology of placental weight increase caused by hyperglycemia.

RNA sequencing and bioinformatics analysis of human lens epithelial cells in age-related cataract.

BACKGROUND: Age-related cataract (ARC) is the main cause of blindness in older individuals but its specific pathogenic mechanism is unclear. This study aimed to identify differentially expressed genes (DEGs) associated with ARC and to improve our understanding of the disease mechanism. METHODS: Anterior capsule samples of the human lens were collected from ARC patients and healthy controls and used for RNA sequencing to detect DEGs. Identified DEGs underwent bioinformatics analyses, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Subsequently, reverse transcription quantitative RT-qPCR was used to validate the different expression levels of selected genes. RESULTS: A total of 698 up-regulated DEGs and 414 down-regulated DEGs were identified in ARC patients compared with controls by transcriptome analysis. Through GO and KEGG bioinformatics analysis, the functions of significantly DEGs and their possible molecular mechanisms were determined. Sequencing results were verified by RT-qPCR as being accurate and reliable. CONCLUSIONS: This study identified several genes associated with ARC, which improves our knowledge of the disease mechanism.

High-expression of ROCK1 modulates the apoptosis of lens epithelial cells in age-related cataracts by targeting p53 gene.

BACKGROUND: Age-related cataract (ARC) is a serious visual impairment disease, and its pathogenesis is unclear. This article aims to investigate the role of ROCK1 in the apoptosis of lens epithelial cells (LECs) in age-related cataracts. METHODS: We collect anterior capsule samples from normal people, patients with age-related cataracts, young mice and naturally aging cataract mice. The oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with H2O2. MTT, Hoechst 33342, and TUNEL assay were performed to explore proliferation and apoptosis. HE assay was used to observe cell morphology. The gene and protein expression were assessed by quantitative real-time PCR, western blot, immunofluorescence, and immunohistochemical staining. RESULT: The results from the clinic and mice experiments showed that the numbers of lens epithelial cells from cataract individuals were less than the control individuals. In vitro, the apoptotic cells were increased in lens epithelial cells under H2O2 treatment. The ROCK1 protein level increased in the lens epithelial cells from age-related cataract patients and the old mice, respectively. Meanwhile, the up-regulation of the ROCK1 gene was associated with H2O2-induced HLE-B3 cells apoptosis. MTT and apoptosis assay showed ROCK1 was necessary in mediating H2O2-induced lens epithelial cells apoptosis through ROCK1 over-expression and knockdown experiment, respectively. Further investigation showed that p53 protein levels had been increased during ROCK1-mediated apoptosis in response to H2O2. Besides, ROCK1 phosphorylated p53 at ser15 to up-regulate its protein level. CONCLUSIONS: This study established the novel association of ROCK1/p53 signaling with lens epithelial cells apoptosis and age-related cataract genesis.

MDM2 phosphorylation mediates H2O2-induced lens epithelial cells apoptosis and age-related cataract.

Lens epithelial cells (LECs) apoptosis induced by oxidative stress is a major factor in age-related-cataract (ARC) pathogenesis, but there are still many blind nodes in this progress. This study aimed to investigate the effects of MDM2 phosphorylation in ARC and H2O2-induced lens epithelial cells apoptosis. Our results confirmed that the levels of p-MDM2 (Ser166) and p-MDM2 (Ser186) in the anterior lens capsules of human cataracts were reduced compared to that in normal capsules. Similarly, in naturally aging cataract mice, the level of MDM2 phosphorylation also decreased. Oxidative stress-induced apoptosis model was constructed by cultivating HLE-B3 cells with 200 µM H2O2. It was confirmed that MDM2 could regulate lens epithelial cell apoptosis, and MDM2 inhibitors could partly inhibited AKT's role in suppressing apoptosis induced by H2O2. Besides, we examed the decreased level of p-AKT(Ser473) in apoptosis of lens epithelial cells and ARC. Our study revealed that MDM2 phosphorylation mediated H2O2-induced lens epithelial cells apoptosis and ARC, which could provide new ideas for the clinical treatment of ARC.

Integrated impacts of turbulent mixing and NOX-O3 photochemistry on reactive pollutant dispersion and intake fraction in shallow and deep street canyons.

We employ computational fluid dynamics (CFD) simulations with NO-NO2-O3 chemistry to investigate the impacts of aspect ratios (H/W = 1,3,5), elevated-building design, wind catchers and two background ozone concentrations ([O3]b = 100/20 ppb) on reactive pollutant dispersion in two-dimensional (2D) street canyons. Personal intake fraction of NO2 (P_IFNO2) and its spatial mean value in entire street (i.e. street intake fraction ) are calculated to quantify pollutant exposure in near-road buildings. Chemical reaction contribution of NO2 exposure (CRC), O3 depletion rate (dozone) and photostationary state defect (δps) are used to analyze the interplay of turbulent and chemical processes. As H/W increases from 1, 3 to 5 with [O3]b = 100 ppb, the flow pattern turns from single-main-vortex structure to two-counter-rotating-vortex structure, and pedestrian-level velocity becomes 1-2 orders smaller. The high-dozone regions and low-|δps| regions get larger with more complete chemical reactions. Consequently, passive  rises 1 order (4.09-5.71 ppm to 41.76 ppm), but reactive only increases several times (17.80-21.28 ppm to 58.50 ppm) and the contribution of chemistry (CRC) decreases with higher H/W. Thus, chemistry raises  more effectively in shallow street canyons (H/W = 1-3). In deep street canyons (H/W = 5), elevated-building design and wind catchers destroy two-counter-rotating-vortex structure, improve street ventilation and reduce passive by 2 and 1 orders (41.76 ppm to 0.38-5.16 ppm), however they only reduce reactive by about 97.5% and 75% (58.50 ppm to 1.61-14.48 ppm). Such building techniques induce lower O3 depletion rate but greater chemical contribution. Finally, raising [O3]b from 20 to 100 ppb causes greater O3 depletion rate and chemical contribution and produces larger . For deep street canyons, the impact of higher [O3]b on is weaker than that in shallow street canyons, while it becomes stronger when fixing elevated-building design and wind catchers. This study provides some innovative findings on reactive pollutant exposure in 2D street canyons and offers effective CFD methodologies to evaluate pollutant exposure with more complicated chemistry and urban configurations.

Numerical evaluations of urban design technique to reduce vehicular personal intake fraction in deep street canyons.

High-rise deep street canyons usually experience poor ventilation and large vehicular pollutant exposure to residents in near-road buildings. Investigations are still required to clarify the flow and dispersion mechanisms in deep street canyons and explore techniques to reduce such large pollutant exposure. By conducting computational fluid dynamics (CFD) simulations validated by wind tunnel data and scale-model outdoor field measurements, we investigate the integrated impacts of aspect ratios, first-floor and second-floor elevated building designs, viaduct settings, height variations and wind catchers on the flow, personal intake fraction (P_IF) of CO (carbon dioxide) and its spatial mean value 〈P_IF〉 in two-dimensional (2D) street canyons. Results show that cases with H/W = 5 experience two counter-rotating vortices, much poorer ventilation and 1-2 orders larger 〈P_IF〉 (43.6-120.8 ppm) than H/W = 1 and 3 (3.8-4.3 and 5.6-5.8 ppm). Moreover, in cases with H/W = 5 the height variation results in three vertically-aligned vortices and much weaker wind, subsequently produces greater 〈P_IF〉 (1402-2047 ppm). To reduce 〈P_IF〉 with H/W = 5, various urban designs are evaluated. The first-floor elevated building design creates more effective ventilation pathways than the second-floor elevated type does and reduces 〈P_IF〉 at H/W = 5 by five orders (1402 to ~0.01 ppm) or two orders (43.6 to ~0.1 ppm) in cases with or without the height variation. However, such reductions at H/W = 1 and 3 are only 76.8%-81.4% and 22.4%-36.2% respectively. Wind catchers destroy the multi-vortex flow pattern as H/W = 5, produce a contra-clockwise main vortex and reduce 〈P_IF〉 by 1-2 orders for cases with or without the height variation.